MCB 110:Biochemistry of the Central Dogma of MB Part 1. DNA replication, repair and genomics (Prof. Alber) Part 2. RNA & protein synthesis. Prof. Zhou Part 3. Membranes, protein secretion, trafficking and signaling Prof. Nogales MCB 110:Biochemistry of the Central Dogma of MB Part 1. DNA replication, repair and genomics (Prof. Alber) Part 2. RNA & protein synthesis. Prof. Zhou Part 3. Membranes, protein secretion, trafficking and signaling Prof. Nogales 1
DNA structure summary 1 1. W & C (1953) modeled average DNA (independent of sequence) as an: anti-parallel, right-handed, double helix with H-bonded base pairs on the inside and the sugar-phosphate backbone on the outside. 2. Each chain runs 5 to 3 (by convention). Profound implications: complementary strands suggested mechanisms of replication, heredity and recognition. Missing Structural variation in DNA as a function of sequence Tools to manipulate and analyze DNA (basis for biotechnology, sequencing, genome analysis) DNA schematic (no chemistry) 1. Nucleotide = sugar-phosphate + base 2. DNA strands are directional 3. Duplex strands are antiparallel and complementary. Backbone outside; H-bonded bases stacked inside. 4. The strands form a double helix 2
Nucleic-acid building blocks nucleoside glycosidic bond nucleotide Geometry of DNA bases and base pairs! C G T A H-bonds satisfied Similar width Similar angle to glycosidic bonds Pseudo-symmetry of 180 rotation 3
Major groove and minor groove definitions Major groove Major groove Opposite the glycosydic bonds Minor groove Minor groove Subtended by the glycosydic bonds Comparison of B DNA and A DNA (formed at different humidity) Bps near helix axis Bps off helix axis 3.4-3.6 Å Major groove (winds around) Minor groove (winds around) bp/turn Base tilt Major groove Minor groove P-P distance 10 small wide Narrow 6.9 Å 11 20 narrow & deep wide & shallow 5.9 Å 4
Average structure of dsrna (like A DNA) 3 side view Bases tilted 5 5 Minor groove shallow and wide Major groove deep and narrow (distortions needed for proteins to contact bases) 3 End view Twist/bp ~32.7 ~11 bp/turn DNA structure and stability DNA structure varies with sequence 1. Dickerson dodecamer crystal structure 2. Twist, roll, propeller twist and displacement 3. Variation in B-DNA and A-DNA Proteins recognize variations in DNA structure DNA stability Depends on sequence & conditions Forces that stabilize DNA: H-bonds, stacking, and interactions with ions and water 5
Crystal structure of the Dickerson dodecamer Experiment -- 1981 Synthesize and purify 12-mer: d(cgcgaattcgcg) = sequence Crystallize Shine X-ray beam through crystal from all angles Record X-ray scattering patterns Calculate electron density distribution Build model into e - density and optimize fit to predict the data Display and analyze model Results B-DNA!! The structure was not a straight regular rod. There were sequence-dependent variations (that could be read out by proteins). Two views of the Dickerson dodecamer 1. Double helix: Anti-parallel strands, bps stacked in the middle 2. Not straight (19 bend/12 bp, 112 Å radius of curvature) 3. Core GAATTC: B-like with 9.8 bp/turn 4. Flanking CGCG more complex, but P-P distance = 6.7 Å (like B) 5. Bps not flat. Propeller twist 11 for GC and 17 for AT 6. Hydration: water, water everywhere on the outside (not shown). 6
Nomenclature for helical parameters Propeller twist: dihedral angle of base planes. Displacement: distance from helix axis to bp center Slide: Translation along the C6-C8 line Twist: relative rotation around helix axis Roll: rotation angle of mean bp plane around C6-C8 line Tilt: rotation of bp plane around pseudo-dyad perpendicular to twist and roll axes Slide Propeller twist, roll and slide No roll or propeller twist Slide = -1 Å to avoid clash * 20 propeller twist Or roll = 20 and slide = + 2Å to promote cross-chain purine stacking 7
Slide and helical twist Slide = translation along the long (C6-C8) axis of the base pair Regular DNA variations B-like A-like 8
Helical parameters of the dodecamer C1/G24 G12/C13 Range 4.9-18.6 32.2-41.4 8.1-11.2 3.14-3.54 Å Helical parameters of the dodecamer C1/G24 G12/C13 Range 4.9-18.6 32.2-41.4 8.1-11.2 3.14-3.54 Å 9
Helical parameters of the dodecamer C1/G24 G12/C13 Range 4.9-18.6 32.2-41.4 8.1-11.2 3.14-3.54 Å Base stacking maximizes favorable interactions Clashes due to propeller twist can be alleviated by positive roll (bottom left) or changes in helical twist (right) N atoms close N atoms separated Δ roll Δ helical twist 10
Different patterns of H-bond donors and acceptors bases in different base pairs (gray) Major groove side (w) Most differences in H-bond donors and acceptors occur in the major groove! Sequence-specific recognition uses major-groove contacts. Minor groove side (S) Seeman, Rosenberg & Rich (1976), Proc Natl Acad Sci USA 73, 804-8. Lac repressor headpiece binds differently to specific and nonspecific DNAs Symmetric operator Natural operator Bent DNA Straight DNA Nonspecific DNA 11
E. coli lac repressor tetramer binds 2 duplexes Hinge helix Headpiece NH 2 N-subdomain C-subdomain Tetramerization helix LacI tetramer E. coli lac repressor tetramer binds 2 duplexes Hinge helix Headpiece NH 2 N-subdomain C-subdomain Tetramerization helix Repressor tetramer loops DNA 12
E. coli catabolite activator protein (CAP) Stabilizes kinks in the DNA Human TATA binding protein binds in the minor groove and stabilizes large bends Twist along the DNA DNA bent 13
Human TATA binding protein binds in the minor groove and stabilizes large bends TBP TBP DNA View into the saddle End view DNA bending by E. coli AlkA DNA glycosylase 66 bend Leu125 inserted into the DNA duplex! 14
Base flipping in DNA repair enzymes Human Alkyl Adenine DNA Glycosylase Phage T4 A Glycosyl Transferase, AGT What causes bases to flip out? 15
What cause bases to flip out? Thermal fluctuations Fluctuations include denaturation Native Denatured + T m = 50/50 native/denatured T 16
Tm depends on? Tm depends on? DNA Length Base composition DNA Sequence Salt concentration Hydrophobic and charged solutes Bound proteins Supercoiling density 17
Length dependence of DNA stability Fraction denatured 10 20 30 No further increase > ~50 base pairs Temperature C Tm depends on G+C content Why? 18
Tm depends on G+C content Why? GC bps contain 3 H-bonds and stack better. Calculated base stacking energies GC best AT worst 19
Tm depends on ionic strength High KCl stabilizes duplex DNA Why? Other conditions that change Tm Mg 2+ ions Stabilize (why?) Polyamines: spermidine and spermine + + + NH 3 -CH 2 -CH 2 -CH 2 -NH 2 -CH 2 -CH 2 -CH 2 -CH 2 -NH 3 } NH 3 -CH 2 -CH 2 -CH 2 -NH 2 -CH 2 -CH 2 -CH 2 -CH 2 -NH 2 -CH 2 CH 2 -CH 2 -NH 3 + + + + DMSO formamide H 3 C CH 3 HC NH 2 C O O } Destabilize (why?) 20
Duplex stability depends on length (to a point) and base composition (GC content) Two formulas for oligonucleotide Tm 1. Tm = (# of A+T) x 2 + (# of G+C) x 4 2. Tm= 64.9 +41 x ((yg+zc-16.4)/ (wa+xt+yg+zc)) where w, x, y, z are the numbers of the respective nucleotides. Summary 1. DNA structure varies with sequence. 2. Propeller twist, helix twist, roll, slide, and displacement (local features) vary in each base step. 3. These differences alter the positions of interacting groups relative to ideal DNA. 4. Structural adjustments maximize stacking. 5. Proteins can read out base sequence directly and indirectly (e.g. H 2 O, PO 4 positions, structure and motions). 6. Proteins can trap transient structures of DNA. 7. Duplex stability varies with sequence, G+C > A+T 8. High salt, Mg 2+, polyamines increase duplex stability. 9. DMSO and formamide decrease duplex stability. 10. Stability increases with oligonucleotide length up to a point. 21
Chemical structures of 4 bases each in DNA and RNA RNA only DNA only DNA and RNA Ribo-AGUC chain Chemical schematic One-letter code Chain is directional. Convention: 5 3. 22
Six backbone dihedral angles (α ζ) per nucleotide Is ssdna floppy or rigid? Two orientations of the bases: Anti and syn 23
Experiment X-rays Fiber diffraction pattern revealed dimensions and helix of B DNA DNA fiber X-ray film Conclusion: Helix with 10 bp/repeat and 3.4 Å between bps Average structure of B DNA Ball-and-stick Space filling side view End view 24
Average structure of B DNA Ball-and-stick Space filling 5 3 side view R-handed Helix Minor groove (narrow) and Major groove (wide) Anti-parallel strands End view Equal twist/bp (36 ) 10 bp/turn side view Bases tilted ~20 5 Average structure of A DNA Ball-and-stick Space filling 5 3 3 Minor groove (shallow and wide) Major groove (deep and narrow) End view Twist/bp ~32.7 ~11 bp/turn 25